This project focuses on signal transduction pathways mediating the cellular response to extracellular stimuli in normal and aged cells. Studies over the past year have concentrated on two topics. (1) Structure/function analysis of MAP kinase phosphatase-1(MKP-1). MAP kinase phosphatases are a group of dual specificity phosphatases induced by extracellular stimuli including growth factors and stress. They play an important role in the feedback control of the MAP kinase cascades. It has been demonstrated that some members of this class of phosphatases can exhibit selectivity toward a specific MAP kinase subfamily(s). The goal of this study is to understand the substrate specificity of MKP-1 and the structural basis responsible for the specificity. We have simultaneously expressed and activated JNK, ERK, and p38 in the same cells, and analyzed their dephosphorylation by MKP- 1. We have found that MKP-1 preferentially inactivates p38> JNK> ERK. By creating numerous MKP-1 truncation mutants and swapping domains between MKP-1 and MKP-3, we have determined that a region of 177 amino acids of MKP-1 spanning the phosphatase domain is sufficient for optimal binding and inactivation of JNK. This region is required but not sufficient for optimal binding and dephosphorylation of p38. In addition, we have identified a novel auto-inhibitory domain in the extreme carboxyl-terminus of MKP-1. Deletion of this domain dramatically augments its phosphatase activity toward both JNK and ERK MAP kinases. These results not only establish p38 and JNK as the physiological targets of MKP-1 but also provide an explanation for why MKP-1 exhibited substantial phosphatase activity towards ERK in the earlier studies by another group. Our study strongly supports that idea that the main function of MKP-1 is feedback control of the JNK and p38 pathways. (2) Age-associated alteration in signaling pathways in rat hepatocytes. Previously we demonstrated that aging is correlated with decreases in both ERK MAP kinase and p70 S6 kinase activities following growth factor treatment. A decline in the activities of both kinases suggests that aged cells may display an alteration in an early upstream event common to these pathways. We have now compared the earliest signaling events that occur in response to EGF stimulation in young and aged hepatocytes. The activities of both MEK and Ras are also attenuated in magnitude and less sustainable in aged cells in comparison to those in the young cells. In young hepatocytes, EGF triggers rapid tyrosine phosphorylation of EGFR and Shc, and enhances EGFR-Shc complex formation. Formation of this signaling complex in response to EGF is significantly reduced in aged cells, although no difference in tyrosine-phosphorylation of either EGFR or Shc is observed. The alteration in the growth factor receptor complexes may contribute to the decline in proliferation capacity in aged cells. - Signal transduction, MAP kinase, EGF receptor, Shc, MKP-1, aging, stress